Albumin is the most abundant plasma protein, generally constituting slightly over one-half of the total protein in mammalian plasma. In the human body, albumin has the important role of regulating the water balance between blood and tissues, and of functioning as a transport molecule for various compounds, such as bilirubin, fatty acids, cortisol, thyroxine and drugs such as sulfonamides and barbiturates, that are only slightly soluble in water. An albumin deficiency can restrict the transport of slightly water soluble materials throughout the body and a deficiency is signaled in an individual by an abnormal accumulation of serous fluid, or edema. Therefore, it is clinically important to determine whether an individual has a deficiency of serum albumin.
Likewise, it is clinically important to determine if an individual is excreting an excess amount of protein. A normal functioning kidney forms urine in an essentially two step process. Blood flows through the glomerulus, or glomerular region of the kidney. The capillary walls of the glomerulus are highly permeable to water and low molecular weight components of the blood plasma. Albumin and other high molecular weight proteins cannot pass through these capillary walls and are essentially filtered out of the urine so that the protein is available for use by the body. The liquid containing the low molecular weight components passes into the tubules, or tubular region, of the kidney where reabsorption of some urine components, such as low molecular weight proteins; secretion of other urine components; and concentration of the urine occurs. As a result, through the combined processes of the glomerulus and tubules the concentration of proteins in urine should be minimal to non-existent. Therefore, abnormally high amounts of albumin and/or low-molecular weight proteins in urine must be detected and related to a physiological dysfunction.
The relatively high concentration of albumin in the urine of an individual usually is indicative of a diseased condition. For example, the average normal concentration of protein in urine varies from about 2 mg/dL to about 8 mg/dL, with approximately one-third of the total urinary protein being serum albumin. However, in a majority of diseased states, urinary protein levels increase appreciably, such that albumin accounts for from about 60 percent to about 90 percent of the excreted protein. The presence of an abnormal increased amount of protein in the urine, known as proteinuria, is one of the most significant indicators of renal disease, and may be indicative of various other non-renal related diseases.
Therefore, in order to determine if an individual has an albumin deficiency and/or to determine if an individual excretes an excess amount of protein, and in order to monitor the course of medical treatment to determine the effectiveness of the treatment, simple, accurate and inexpensive protein detection assays have been developed. Furthermore, of the several different assay methods developed for the detection or measurement of protein in urine and serum, the methods based on dye binding techniques have proven especially useful because dye binding methods are readily automated and provide reproducible and accurate results.
In general, dye binding techniques utilize pH indicator dyes that are capable of interacting with a protein, such as albumin, and that are capable of changing color upon interaction with a protein absent any change in pH. When a pH indicator dye interacts with, or binds to, a protein, the apparent pK.sub.a (acid dissociation constant) of the indicator dye is altered and the dye undergoes a color transition, producing the so-called "protein-error" phenomenon. In methods utilizing the dye binding technique, an appropriate buffer maintains the pH indicator dye at a constant pH to prevent a color transition of the pH indicator dye due to a substantial shift in pH. Due to the "protein-error" phenomena, upon interaction with the protein, the pH indicator dye undergoes a color transition that is identical to the color change arising because of a change in the pH. Examples of pH indicator dyes used in the dry phase assay of proteins that are capable of interacting with or binding to proteins and exhibiting "protein-error" color transitions include tetrabromophenol blue and tetrachlorophenol-3,4,5,6-tetrabromosulfophthalein.
Although pH indicator dyes have been used extensively in protein assays, several disadvantages still exist in protein assay methods utilizing indicator dyes. For example, methods based upon pH indicator dyes either cannot detect or cannot quantitatively differentiate between protein concentrations below approximately 15 mg/dL. In addition, although several simple semiquantitative tests and several complex quantitative tests are available for the determination of the total protein content in a test sample, the majority of these assay methods, with the notable exception of the simple colorimetric reagent test strip, require the precipitation of protein to make quantitative protein determinations.
The colorimetric reagent test strip utilizes the previously discussed ability of proteins to interact with certain acid-base indicators and to alter the color of the indicator without any change in the pH. For example, when the indicator tetrabromophenol blue is buffered to maintain a constant pH of approximately 3, the indicator imparts a yellow color to solutions that do not contain protein. However, for solutions containing protein, the presence of protein causes the buffered dye to impart either a green color or a blue color to solution, depending upon the concentration of protein in the solution. Consequently, the development of a green background color in the carrier matrix of a dry phase test strip can interfere in the assay for proteins.
Some colorimetric test strips used in protein assays have a single test area consisting of a small square pad of a carrier matrix impregnated with a buffered pH indicator dye, such as tetrabromophenol blue. Other colorimetric test strips are multideterminant reagent strips that include one test area, or test pad, for protein assay as described above, and further include several additional test pads on the same strip to permit the simultaneous assay of other urinary constituents, like pH. For both types of colorimetric test strips, the assay for protein in urine is performed simply by dipping the colorimetric test strip into a well mixed, uncentrifuged urine sample, then comparing the resulting color of the test pad of the test strip to a standardized color chart provided on the colorimetric test strip bottle.
As will be discussed more fully hereinafter, in a multideterminant reagent strip, the protein test pad often is positioned adjacent to the pH test pad. In addition, the reagents incorporated into the protein test pad, buffered at a pH of about 3 to about 4, often runover from the protein test pad onto the pH test pad when the multideterminant test strip is dipped into the urine sample. Consequently, the pH assay is inaccurate because of contamination by the acidic indicator reagent composition used in the protein assay. Surprisingly and unexpectedly, the test device and method of the present invention also essentially eliminate the problem of indicator reagent composition runover.
For test strips utilizing tetrabromophenol blue, buffered at pH 3, as the indicator dye, semiquantitative assays for protein can be performed and are reported as negative, trace, or one "plus" to four "plus". A negative reading, or yellow color, indicates that the urine contains no protein, as demonstrated by the lack of a color transition of the indicator dye. A trace reading may indicate from about 5 to about 20 mg/dL of protein in the urine. The one "plus" to four "plus" readings, signified by color transitions of green through increasingly dark shades of blue, are approximately equivalent to urine protein concentrations of 30, 100, 300, and over 2000 mg/dL, respectively, and serve as reliable indicators of increasingly severe proteinuria. Therefore, eliminating the development of an interfering green background color in the carrier matrix is important for an accurate protein analysis. The elimination of the green background color is especially important when the test sample includes 30 mg/dL or less of protein, because at such a low protein concentration range, the test pad responds to the protein content by exhibiting a green or yellow color.
In accordance with the above-described method, an individual can readily determine, visually, that the protein content of a urine sample is in the range of 0 mg/dL to about 30 mg/dL. However, the color differentiation afforded by the presently available commercial test strips is insufficient to allow an accurate determination of protein content in urine between 0 mg/dL and about 15 mg/dL. The inability to detect and differentiate between low protein concentrations is important clinically because a healthy person usually has a urine protein level in the range of about 10 mg/dL to about 20 mg/dL. Therefore, it could be clinically important to know more precisely the urine protein content of an individual, rather than merely estimating the protein content at some value less than about 30 mg/dL.
Of course, the protein content of a urine sample can be determined more precisely by semiquantitative protein precipitation techniques or by quantitative 24 hour protein precipitation techniques. However, these tests are time consuming and relatively expensive. Furthermore, the precipitation tests must be run in a laboratory by trained personnel, and therefore are unavailable for the patient to perform at home to quickly determine urine protein content and to monitor the success or failure of a particular medical treatment.
Therefore, it would be extremely advantageous to have a simple, accurate and trustworthy method of assaying urine for protein content that allows visual differentiation of protein levels in the ranges of 0 mg/dL to about 10 mg/dL, about 10 mg/dL to about 20 mg/dL, and about 20 mg/dL to about 30 mg/dL, and upwards to between about 100 mg/dL to about 300 mg/dL. By providing such an accurate method of determining urine protein concentration in an easy to use form, such as a dip-and-read test strip, the urine assay can be performed by laboratory personnel to afford immediate test results, such that a diagnosis can be made without having to wait up to one day for assay results and medical treatment can be commenced immediately. In addition, the test strip method can be performed by the patient at home to more precisely monitor low levels of protein in urine and/or the success of the medical treatment the patient is undergoing. Finally, the method and test device used in a protein assay should not adversely affect or interfere with other test pads that are present on a multideterminant test strip.
As will be described more fully hereinafter, the method of the present invention allows the fast, accurate and trustworthy protein assay of urine by utilizing a test strip that includes a test pad comprising a new and improved carrier matrix incorporating an indicator reagent composition. The carrier matrix comprises a fibrous, bibulous substrate, such as filter paper, homogeneously impregnated with a polymerized urethane-based compound. Surprisingly and unexpectedly, the carrier matrix of the present invention essentially eliminates the interfering green background color that can develop on dry phase test strips. The new and improved carrier matrix of the present invention, by essentially eliminating the development of an interfering green background color, enhances the visual color resolution, and therefore the sensitivity, of the assay, thereby allowing urine protein concentrations to be accurately determined at levels of approximately 30 mg/dL or less. In addition, the carrier matrix also essentially eliminates the runover of the protein indicator reagent composition onto adjacent test pads of a multideterminant test strip, thereby improving the accuracy of assays performed by the adjacent test pad, like a pH assay.
In addition, the test device and method of the present invention can be used to determine the presence or concentration of low molecular weight proteins, such as Bence Jones proteins, in a test sample. All prior art assay techniques for low molecular weight proteins involve either immunoelectrophoresis or heat test methods that are time consuming, relatively expensive and are not amenable for use by an individual at home to detect low molecular weight proteins in urine.
Bence Jones proteins belong to a class of urinary proteins having a low molecular weight of approximately 20,000 and that are small enough to pass through the glomerular filters of the kidney. However, the Bence Jones proteins usually are reabsorbed in the tubular section of the kidney. Therefore, the concentration of Bence Jones proteins is negligible in the urine of a healthy person. As a result, a significant amount of Bence Jones proteins in urine generally is clinically significant. Overall, the detection and measurement of the concentration of low molecular weight proteins in urine is important because certain diseases are characterized by the excretion of specific low molecular weight proteins (globulins) rather than by diffuse proteinuria characterized by elevated albumin levels.
For example, the Bence Jones proteins represent a portion of the high molecular weight plasma myeloma globulin, and therefore are found in increased amounts in the urine of more than one-half of the patients suffering from multiple myeloma or leukemia. Bence Jones proteinuria also is found in the urine of many patients suffering from macroglobulinemia and primary systemic amyloidosis. In addition, an increased excretion of a specific globulin that is similar to Bence Jones proteins occurs in Franklin's disease; and patients with renal tubular disorders, such as the Fanconi syndrome, show a substantial increase in the quantity of globulins excreted in the urine. Accordingly, investigators have searched for a simple assay for low molecular weight proteins because the dye-binding method used in presently available commercial test strips is insensitive to low molecular weight proteins, like Bence Jones proteins. Surprisingly and unexpectedly, the method of the present invention provides an improved technique to detect and measure the concentration of low molecular weight proteins, like Bence Jones proteins, by incorporating an indicator reagent composition into a new and improved carrier matrix comprising a fibrous, bibulous substrate homogeneously impregnated with a polymerized urethane-based compound, that effectively resists the development of an interfering green background color.
The Bence Jones proteins differ from all other urinary proteins in that they coagulate upon heating to temperatures between about 45.degree. C. and about 60.degree. C., and then redissolve on further heating to the boiling point of test sample. This peculiar characteristic of Bence Jones proteins has been the basis of all qualitative and semiquantitative determinations for Bence Jones proteins. The dye binding technique used in commercially available test strips has proved insensitive to Bence Jones proteins because the much greater relative concentration of higher molecular weight proteins, such as albumin, in the urine effectively interferes with and masks the presence of Bence Jones proteins. Furthermore, it is inconvenient and costly to separate the albumin from Bence Jones proteins, thereby negating the utility of separating the albumin from the Bence Jones proteins before using a dry phase test strip.
Presently, dry phase test strips are capable of only marginally detecting the presence of Bence Jones proteins in urine. However, incorporating an indicator reagent composition into the improved carrier matrix of the present invention provides an improved assay test pad for the detection of Bence Jones proteins in liquid test samples, such as urine. Overall, the non-greening carrier matrix of the present invention provides improved color resolution of a color transition resulting upon contact of a protein-containing test sample with the indicator reagent composition, therefore improving assay sensitivity and allowing the detection and measurement of proteins, like albumin, in liquid test samples to levels as low as 10 mg/dL.
Proteinuria resulting either from abnormally high albumin levels or the presence of low molecular weight proteins depends upon the precise nature of the clinical and pathological disorder and upon the severity of the specific disease. Proteinuria can be intermittent or continuous, with transient, intermittent proteinuria usually being caused by physiologic or functional conditions rather than by renal disorders. Therefore, accurate assays of urine and other test samples for protein must be available for both laboratory and home use. The assays must permit the detection or measurement of the proteins of interest, either albumin and/or Bence Jones proteins, such that a correct diagnosis can be made and correct medical treatment implemented, monitored and maintained. In addition, it would be advantageous if the protein assay method, both for high molecular weight proteins, like albumin, and low molecular weight proteins, like Bence Jones proteins, could be utilized in a dip-and-read format for the easy and economical, qualitative or quantitative determination of protein in urine or other test samples.
Furthermore, any method of assaying for protein in urine or other test samples must yield accurate, trustworthy and reproducible results by utilizing a method that provides a detectable or measurable color transition as a result of an interaction between the indicator reagent composition and the protein, and not as a result of a competing chemical or physical interaction, such as a pH change or preferential interaction with a test sample component other than protein. Moreover, it would be advantageous if the protein assay method is suitable for use in dry reagent strips for the rapid, economical and accurate determination of protein in urine and other test samples. Additionally, the method and test pad, comprising the carrier matrix and the indicator reagent composition, utilized in the assay for protein, and the indicator reagent composition, should not adversely affect or interfere with the other test reagent pads that are present on multideterminant test pad strips.
Prior to the present invention, no known method of assaying urine or other test samples for proteins utilized a test device including a test pad comprising a non-greening carrier matrix, comprising a fibrous, bibulous substrate homogeneously impregnated with a polymerized urethane-based compound. The carrier matrix provides improved color resolution and increased assay sensitivity compared to test pads absent the homogeneous impregnation of the polymerized urethane-based compound, such that accurate and trustworthy protein assays can be made for protein concentrations of about 30 mg/dL and below. Furthermore, no other test device includes a test pad that so effectively resists the runover, or bleeding, of the indicator reagent composition onto adjacent test pads that interferes with the assay performed by the adjacent test pad.
In addition, although a dry phase chemistry test strip utilizing a dye, such as tetrabromophenol blue or tetrachlorophenol-3,4,5,6-tetrabromosulfonephthalein, has been used extensively for several years, no dry phase test strip has utilized a test pad comprising a non-greening and non-bleeding carrier matrix comprising a fibrous, bibulous substrate, such as filter paper, homogeneously impregnated with a polymerized urethane-based compound. The non-greening carrier matrix is manufactured by a method that essentially eliminates the development of an interfering green background color of the carrier matrix, therefore improving visual color resolution and increasing assay sensitivity, especially at lower protein concentration levels. Furthermore, until the method of the present invention, dry phase test strip procedures were available principally to test for total protein concentration, i.e., for albumin, only down to levels as low as 30 mg/dL. However, surprisingly and unexpectedly, because of the essential elimination of the interfering green background color, the method of the present invention facilitates the dry phase test strip assay of urine and other test samples for albumin down to levels as low as 10 mg/dL, and for the presence of low molecular weight proteins, such as Bence Jones proteins. Moreover, the carrier matrix of the present invention essentially eliminates the indicator reagent composition from bleeding onto adjacent test pads and interfereing with the adjacent test pad assay.
The prior art contains numerous references on the wet phase and the dry phase chemistry utilized in the pH indicator dye method of assaying urine for proteins. For example, Keston U.S. Pat. No. 3,485,587 discloses the basic dye binding technique used to assay for proteins at a constant pH. Keston teaches utilizing a single indicator dye, maintained at a constant pH slightly below the pK.sub.a (acid dissociation constant) of the dye and impregnated into a dry test paper, like filter paper, to determine the presence or concentration of albumin by monitoring the color transition of the dye. Free, et al., in U.S. Pat. No. 3,095,277, also discloses a method of detecting the albumin content of liquid test samples by incorporating a suitable indicator composition into a bibulous carrier, like untreated filter paper. Similarly, Atkinson et al in U.S. Pat. No. 3,438,737 discloses a test device comprising a test composition impregnated into an untreated bibulous matrix, such as filter paper, wood strips, synthetic plastic fibrous materials, non-woven fabrics and woven fabrics for detecting protein in fluids.
Some investigators have treated a fibrous, bibulous substrate, like paper, with a polyurethane. For example, Isgur et al, in G.B. 2,068,034, disclosed treating paper with a polyurethane polymer amine salt, then curing the polyurethane polymer amine salt at from 104.degree. C. to 150.degree. C. The treatment with the polyurethane polymer amine salt increased the wet strength of paper, cartons, cardboard and related products. Similarly, Daude et al, in EP 17598 disclosed a method of improving the mechanical properties of paper, cardboard and related products, either in the wet stage or the dry stage, by impregnating the paper product with an aqueous emulsion including a blocked polyurethane prepolymer and a deblocking catalyst. The impregnated paper product then is heated at from 150.degree. C. to 350.degree. C. for 0.5 sec. to 6 sec. to deblock the polyurethane prepolymer. In addition, Dahl in U.S. Pat. No. 3,702,781, disclosed a process of impregnating paper with certain polyurethane polymers to strengthen the dry and the wet tensile strength of paper products, and to increase the delamination resistance of paper without a loss of paper flexibility. The polyurethane impregnation was performed by heating a permeable paper product having incorporated therein a composition including a polyisocyanate, a polyol, and a catalyst in an inert solvent.
However, the above-cited references do not teach or suggest, either alone or in combination, that a polyurethane-treated fibrous, bibulous substrate can be used in a diagnostic device to permit a more accurate determination of the amount of an analyte, like protein, and especially low amounts of an analyte, in a test sample. The references also do not teach or suggest, alone or in combination, that a polyurethane-treated fibrous, bibulous substrate effectively eliminates the development of a green background color in the treated substrate or eliminates the bleeding of composition incorporated into the polyurethane-treated fibrous, bibulous substrate from the treated substrate upon contact of the polyurethane-treated substrate with a liquid test sample.
In contrast to the prior art, and in contrast to the presently available commercial test strips, the method of the present invention provides increased sensitivity in the detection and measurement of proteins in a liquid test sample, such as a biological fluid, like urine. Surprisingly and unexpectedly, by utilizing a carrier matrix, comprising a fibrous, bibulous substrate homogeneously impregnated with a polymerized urethane-based compound, that effectively resists the development of a green background color in a dry phase test strip, protein levels of about 30 mg/dL and below can be determined accurately. In addition, the method of the present invention also allows the simple and essentially immediate detection of Bence Jones proteins. Hence, in accordance with the method of the present invention, new and unexpected results are achieved in the dry phase reagent strip assay of urine and other test samples for proteins by utilizing a test pad, comprising an indicator reagent composition incorporated into a non-greening carrier matrix, comprising a fibrous, bibulous substrate homogeneously impregnated with a polymerized urethane-based compound, that effectively resists the development of an interfering green background color. Furthermore, the test device and method of the present invention eliminate assay interferences in multideterminant test strips by eliminating indicator reagent composition runover onto adjacent test pads of the multideterminant test strip.